This invention relates to dynamic automatic focusing while scanning a surface and more particularly to a dynamic autofocus control system that rejects discontinuities such as inclusions and edges in the reflectivity of the surface being scanned thereby avoiding transient errors on the parts of the surface that are of interest.
In laser fluorescence scanning of nucleic acid arrays, depth discrimination is required to eliminate fluorescence (and scattering) from materials other than the surface-bound nucleic acids, and a uniform transfer function (illumination to emission sensitivity) across the scanned surface is also desirable. Some optical surface scanning applications require depth discrimination comparable to the expected flatness of the surface to be scanned. Stringent depth discrimination, however, tends to result in brightness non-uniformity unless the surface being scanned is flat compared to the depth discrimination length.
Most laser fluorescence scanners in the field of fluorescence scanning of nucleic acid arrays are of the fixed focus variety. As a result, these scanners must trade depth discrimination for brightness uniformity. The designers of fixed focus scanners generally choose lower depth discrimination (xe2x89xa7100 microns), effectively reducing sensitivity of their instruments by allowing more spurious background signal into the measurement. Some scanners (e.g., the Hewlett-Packard GeneArray(copyright) Scanner) use a static focus mechanism. Static focus assumes that the surface of interest is planar, measures focus at three points of the surface, and aligns the surface to minimize focus error at these three points. This approach is clearly immune to transient effects while scanning because it makes no attempt to focus while scanning. On the other hand, such a system does not focus optimally on surfaces that are not flat. Typical glass microscope slides have commonly observed deviations from perfect flatness on the order of 10 microns peak-to-peak rising to 50-100 microns when inserted into a mechanical holder or cartridge under scanning conditions. The design goal for depth discrimination in high performance scanners of biological materials is typically below 50 microns.
In another scanning context, compact disk (CD) players use a dynamic focusing servo system that measures reflectivity of the surface to position the scan lens. However, scratches and surface imperfections can cause transient effects that drive the focus system away from the desired operating point. CD players compensate for some level of surface irregularities by incorporating large amounts of redundant data into the CD, and by dispersing local data across the surface so that point defects do not affect consecutive bits of the data stream. CD""s have the added advantage that they are round and so do not require the laser beam to scan beyond the surface as is required when scanning a rectangular slide in a reciprocating or raster motion which is used in the scanning of biological materials.
Thus, a problem in any practical realization of dynamic autofocus by reflection is that the surface is never perfect; dirt and inclusions (bubbles, more dirt) change the reflectivity of the surface in bursts. Further, the surface to be scanned may be smaller than the travel of the illuminating beam so that the focus signal is discontinuous at the edge and ill-behaved beyond it. In either case, transient signals are fed through the focus servo system and disturb its response for some time afterwards, propagating focus errors to other parts of the surface which may themselves be flat and clean. It is therefore desirable to focus dynamically while executing a scan over a surface with discontinuous reflectivity such as in raster scanning wherein the scan profile crosses the bounds of the surface, or scanning a surface with scratches, dust, dirt, or inclusions.
In one aspect, the dynamic focus control of the invention includes a lens system having a focal plane and a scanning surface for receiving light from the lens. A position sensor generates a position signal and a focus transducer alters the relative positions of the focal plane and the surface. An inner control loop is responsive to the position signal to drive the transducer and an outer control loop responds to deviation in the relative position of the scanning surface with respect to the focal plane of the lens from a focus setpoint to generate an error signal. The error signal is used to form a position setpoint for the inner control loop. A sample-and-hold functional element is disposed between the inner and outer control loops and is switched by an edge or defect detector.
In a preferred embodiment, the outer control loop has a time constant in the range of three to ten times longer than that of the inner control loop. It is also preferred that the sample-and-hold element provide a gradual state transition such as a timed xe2x80x9cdissolvexe2x80x9d in which the output is equal to the xe2x80x9cheldxe2x80x9d input during the hold state and smoothly changes to equal the xe2x80x9csamplexe2x80x9d input sometime after the transition from hold state to sample state. In another embodiment, the sample-and-hold element delays transition from hold mode to focus mode until the focus loop position setpoint is within a selected tolerance of the held position. A comparator may be provided for receiving expected position of an edge and actual scan position to clock transition from focus mode to position mode. Surface reflectivity may be used to sense an edge or an edge may be sensed from rapid changes in reflectivity alone or in combination with reflectivity itself. The edge or defect detector may also sense a surface property correlated with the presence of inclusions, dirt or scratches. In yet another embodiment, the edge or defect detector employs pre-scanning of the surface to form a map of inclusions which can be used to reject errors by feeding this information forward to a controller during scanning.